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Background: Anthracyclines such as doxorubicin (Dox) are highly effective anti-tumor agents, but their use is limited by dose-dependent cardiomyopathy and heart failure. Our laboratory previously reported that induction of cytochrome P450 family 1 (Cyp1) enzymes contributes to acute Dox cardiotoxicity in zebrafish and in mice, and that potent Cyp1 inhibitors prevent cardiotoxicity. However, the role of Cyp1 enzymes in chronic Dox cardiomyopathy, as well as the mechanisms underlying cardioprotection associated with Cyp1 inhibition, have not been fully elucidated. Methods: The Cyp1 pathway was evaluated using a small molecule Cyp1 inhibitor in wild-type (WT) mice, or Cyp1-null mice ( Cyp1a1/1a2 -/- , Cyp1b1 -/- , and Cyp1a1/1a2/1b1 -/- ). Low-dose Dox was administered by serial intraperitoneal or intravenous injections, respectively. Expression of Cyp1 isoforms was measured by RT-qPCR, and myocardial tissue was isolated from the left ventricle for RNA sequencing. Cardiac function was evaluated by transthoracic echocardiography. Results: In WT mice, Dox treatment was associated with a decrease in Cyp1a2 and increase in Cyp1b1 expression in the heart and in the liver. Co-treatment of WT mice with Dox and the novel Cyp1 inhibitor YW-130 protected against cardiac dysfunction compared to Dox treatment alone. Cyp1a1/1a2 -/- and Cyp1a1/1a2/1b1 -/- mice were protected from Dox cardiomyopathy compared to WT mice. Male, but not female, Cyp1b1 -/- mice had increased cardiac dysfunction following Dox treatment compared to WT mice. RNA sequencing of myocardial tissue showed upregulation of Fundc1 and downregulation of Ccl21c in Cyp1a1/1a2 -/- mice treated with Dox, implicating changes in mitophagy and chemokine-mediated inflammation as possible mechanisms of Cyp1a-mediated cardioprotection. Conclusions: Taken together, this study highlights the potential therapeutic value of Cyp1a inhibition in mitigating anthracycline cardiomyopathy.
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Lysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine towards maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple pathological conditions, including cancer. Measuring sirtuins' activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed 'SIRTify', a high-sensitivity assay for measuring sirtuin activity in vitro and in vivo. SIRTify is based on a split-version of the NanoLuc® luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine acylations and confirm the effects of sirtuin modulators. SIRTify effectively quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications.
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DISC1 is a genetic risk factor for multiple psychiatric disorders. Compared to the dozens of murine Disc1 models, there is a paucity of zebrafish disc1 models-an organism amenable to high-throughput experimentation. We conducted the longitudinal neurobehavioral analysis of disc1 mutant zebrafish across key stages of life. During early developmental stages, disc1 mutants exhibited abrogated behavioral responses to sensory stimuli across multiple testing platforms. Moreover, during exposure to an acoustic sensory stimulus, loss of disc1 resulted in the abnormal activation of neurons in the pallium, cerebellum, and tectum-anatomical sites involved in the integration of sensory perception and motor control. In adulthood, disc1 mutants exhibited sexually dimorphic reduction in anxiogenic behavior in novel paradigms. Together, these findings implicate disc1 in sensorimotor processes and the genesis of anxiogenic behaviors, which could be exploited for the development of novel treatments in addition to investigating the biology of sensorimotor transformation in the context of disc1 deletion.
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Sphingolipidoses are a subcategory of lysosomal storage diseases (LSDs) caused by mutations in enzymes of the sphingolipid catabolic pathway. Like many LSDs, neurological involvement in sphingolipidoses leads to early mortality with limited treatment options. Given the role of myelin loss as a major contributor toward LSD-associated neurodegeneration, we investigated the pathways contributing to demyelination in a CRISPR-Cas9-generated zebrafish model of combined saposin (psap) deficiency. psap knockout (KO) zebrafish recapitulated major LSD pathologies, including reduced lifespan, reduced lipid storage, impaired locomotion and severe myelin loss; loss of myelin basic protein a (mbpa) mRNA was progressive, with no changes in additional markers of oligodendrocyte differentiation. Brain transcriptomics revealed dysregulated mTORC1 signaling and elevated neuroinflammation, where increased proinflammatory cytokine expression preceded and mTORC1 signaling changes followed mbpa loss. We examined pharmacological and genetic rescue strategies via water tank administration of the multiple sclerosis drug monomethylfumarate (MMF), and crossing the psap KO line into an acid sphingomyelinase (smpd1) deficiency model. smpd1 mutagenesis, but not MMF treatment, prolonged lifespan in psap KO zebrafish, highlighting the modulation of acid sphingomyelinase activity as a potential path toward sphingolipidosis treatment.
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Doenças por Armazenamento dos Lisossomos , Esfingolipidoses , Animais , Esfingomielina Fosfodiesterase/genética , Peixe-Zebra/metabolismo , Saposinas/genética , Alvo Mecanístico do Complexo 1 de RapamicinaRESUMO
The zebrafish is a powerful model system for studying animal development, for modeling genetic diseases, and for large-scale in vivo functional genetics. Because of its ease of use and its high efficiency in targeted gene perturbation, CRISPR-Cas9 has recently gained prominence as the tool of choice for genetic manipulation in zebrafish. However, scaling up the technique for high-throughput in vivo functional genetics has been a challenge. We recently developed a method, Multiplexed Intermixed CRISPR Droplets (MIC-Drop), that makes large-scale CRISPR screening in zebrafish possible. Here, we outline the step-by-step protocol for performing functional genetic screens in zebrafish by using MIC-Drop. MIC-Drop uses multiplexed single-guide RNAs to generate biallelic mutations in injected zebrafish embryos, allowing genetic screens to be performed in F0 animals. Combining microfluidics and DNA barcoding enables simultaneous targeting of tens to hundreds of genes from a single injection needle, while also enabling retrospective and rapid identification of the genotype responsible for an observed phenotype. The primary target audiences for MIC-Drop are developmental biologists, zebrafish geneticists, and researchers interested in performing in vivo functional genetic screens in a vertebrate model system. MIC-Drop will also prove useful in the hands of chemical biologists seeking to identify targets of small molecules that cause phenotypic changes in zebrafish. By using MIC-Drop, a typical screen of 100 genes can be conducted within 2-3 weeks by a single user.
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Sistemas CRISPR-Cas , Peixe-Zebra , Animais , Peixe-Zebra/genética , Sistemas CRISPR-Cas/genética , Estudos Retrospectivos , Testes Genéticos , FenótipoRESUMO
It has been a major challenge to systematically evaluate and compare how pharmacological perturbations influence social behavioral outcomes. Although some pharmacological agents are known to alter social behavior, precise description and quantification of such effects have proven difficult. We developed a scalable social behavioral assay for zebrafish named ZeChat based on unsupervised deep learning to characterize sociality at high resolution. High-dimensional and dynamic social behavioral phenotypes are automatically classified using this method. By screening a neuroactive compound library, we found that different classes of chemicals evoke distinct patterns of social behavioral fingerprints. By examining these patterns, we discovered that dopamine D3 agonists possess a social stimulative effect on zebrafish. The D3 agonists pramipexole, piribedil, and 7-hydroxy-DPAT-HBr rescued social deficits in a valproic-acid-induced zebrafish autism model. The ZeChat platform provides a promising approach for dissecting the pharmacology of social behavior and discovering novel social-modulatory compounds.
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Aprendizado Profundo , Agonistas de Dopamina , Ratos , Animais , Agonistas de Dopamina/farmacologia , Peixe-Zebra , Dopamina , Ratos Sprague-Dawley , Comportamento SocialRESUMO
Pharmacology and toxicology are part of a much broader effort to understand the relationship between chemistry and biology. While biomedicine has necessarily focused on specific cases, typically of direct human relevance, there are real advantages in pursuing more systematic approaches to characterizing how health and disease are influenced by small molecules and other interventions. In this context, the zebrafish is now established as the representative screenable vertebrate and, through ongoing advances in the available scale of genome editing and automated phenotyping, is beginning to address systems-level solutions to some biomedical problems. The addition of broader efforts to integrate information content across preclinical model organisms and the incorporation of rigorous analytics, including closed-loop deep learning, will facilitate efforts to create systems pharmacology and toxicology with the ability to continuously optimize chemical biological interactions around societal needs. In this review, we outline progress toward this goal.
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Toxicologia , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/genética , Farmacologia em RedeRESUMO
Cyanide-a fast-acting poison-is easy to obtain given its widespread use in manufacturing industries. It is a high-threat chemical agent that poses a risk of occupational exposure in addition to being a terrorist agent. FDA-approved cyanide antidotes must be given intravenously, which is not practical in a mass casualty setting due to the time and skill required to obtain intravenous access. Glyoxylate is an endogenous metabolite that binds cyanide and reverses cyanide-induced redox imbalances independent of chelation. Efficacy and biochemical mechanistic studies in an FDA-approved preclinical animal model have not been reported. Therefore, in a swine model of cyanide poisoning, we evaluated the efficacy of intramuscular glyoxylate on clinical, metabolic, and biochemical endpoints. Animals were instrumented for continuous hemodynamic monitoring and infused with potassium cyanide. Following cyanide-induced apnea, saline control or glyoxylate was administered intramuscularly. Throughout the study, serial blood samples were collected for pharmacokinetic, metabolite, and biochemical studies, in addition, vital signs, hemodynamic parameters, and laboratory values were measured. Survival in glyoxylate-treated animals was 83% compared with 12% in saline-treated control animals (p < .01). Glyoxylate treatment improved physiological parameters including pulse oximetry, arterial oxygenation, respiration, and pH. In addition, levels of citric acid cycle metabolites returned to baseline levels by the end of the study. Moreover, glyoxylate exerted distinct effects on redox balance as compared with a cyanide-chelating countermeasure. In our preclinical swine model of lethal cyanide poisoning, intramuscular administration of the endogenous metabolite glyoxylate improved survival and clinical outcomes, and ameliorated the biochemical effects of cyanide.
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Cianetos , Intoxicação , Suínos , Animais , Cianetos/toxicidade , Modelos Animais de Doenças , Antídotos/farmacologia , Antídotos/uso terapêutico , Hemodinâmica , Glioxilatos/uso terapêutico , Intoxicação/tratamento farmacológicoRESUMO
Little is understood about the embryonic development of sociality. We screened 1120 known drugs and found that embryonic inhibition of topoisomerase IIα (Top2a) resulted in lasting social deficits in zebrafish. In mice, prenatal Top2 inhibition caused defects in social interaction and communication, which are behaviors that relate to core symptoms of autism. Mutation of Top2a in zebrafish caused down-regulation of a set of genes highly enriched for genes associated with autism in humans. Both the Top2a-regulated and autism-associated gene sets have binding sites for polycomb repressive complex 2 (PRC2), a regulatory complex responsible for H3K27 trimethylation (H3K27me3). Moreover, both gene sets are highly enriched for H3K27me3. Inhibition of the PRC2 component Ezh2 rescued social deficits caused by Top2 inhibition. Therefore, Top2a is a key component of an evolutionarily conserved pathway that promotes the development of social behavior through PRC2 and H3K27me3.
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NGLY1 deficiency, a rare disease with no effective treatment, is caused by autosomal recessive, loss-of-function mutations in the N-glycanase 1 (NGLY1) gene and is characterized by global developmental delay, hypotonia, alacrima, and seizures. We used a Drosophila model of NGLY1 deficiency to conduct an in vivo, unbiased, small molecule, repurposing screen of FDA-approved drugs to identify therapeutic compounds. Seventeen molecules partially rescued lethality in a patient-specific NGLY1 deficiency model, including multiple serotonin and dopamine modulators. Exclusive dNGLY1 expression in serotonin and dopamine neurons, in an otherwise dNGLY1 deficient fly, was sufficient to partially rescue lethality. Further, genetic modifier and transcriptomic data supports the importance of serotonin signaling in NGLY1 deficiency. Connectivity Map analysis identified glycogen synthase kinase 3 (GSK3) inhibition as a potential therapeutic mechanism for NGLY1 deficiency, which we experimentally validated with TWS119, lithium, and GSK3 knockdown. Strikingly, GSK3 inhibitors and a serotonin modulator rescued size defects in dNGLY1 deficient larvae upon proteasome inhibition, suggesting that these compounds act through NRF1, a transcription factor that is regulated by NGLY1 and regulates proteasome expression. This study reveals the importance of the serotonin pathway in NGLY1 deficiency, and serotonin modulators or GSK3 inhibitors may be effective therapeutics for this rare disease.
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Reposicionamento de Medicamentos , Quinase 3 da Glicogênio Sintase , Animais , Defeitos Congênitos da Glicosilação , Drosophila/genética , Drosophila/metabolismo , Humanos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Complexo de Endopeptidases do Proteassoma/metabolismo , Doenças Raras , Serotonina/genéticaRESUMO
The global crisis of opioid overdose fatalities has led to an urgent search to discover the neurobiological mechanisms of opioid use disorder (OUD). A driving force for OUD is the dysphoric and emotionally painful state (hyperkatifeia) that is produced during acute and protracted opioid withdrawal. Here, we explored a mechanistic role for extrahypothalamic stress systems in driving opioid addiction. We found that glucocorticoid receptor (GR) antagonism with mifepristone reduced opioid addiction-like behaviors in rats and zebrafish of both sexes and decreased the firing of corticotropin-releasing factor neurons in the rat amygdala (i.e., a marker of brain stress system activation). In support of the hypothesized role of glucocorticoid transcriptional regulation of extrahypothalamic GRs in addiction-like behavior, an intra-amygdala infusion of an antisense oligonucleotide that blocked GR transcriptional activity reduced addiction-like behaviors. Finally, we identified transcriptional adaptations of GR signaling in the amygdala of humans with OUD. Thus, GRs, their coregulators, and downstream systems may represent viable therapeutic targets to treat the "stress side" of OUD.
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Transtornos Relacionados ao Uso de Opioides , Síndrome de Abstinência a Substâncias , Corticosteroides , Animais , Hormônio Liberador da Corticotropina , Ratos , Peixe-ZebraRESUMO
Although cyanide's biological effects are pleiotropic, its most obvious effects are as a metabolic poison. Cyanide potently inhibits cytochrome c oxidase and potentially other metabolic enzymes, thereby unleashing a cascade of metabolic perturbations that are believed to cause lethality. From systematic screens of human metabolites using a zebrafish model of cyanide toxicity, we have identified the TCA-derived small molecule glyoxylate as a potential cyanide countermeasure. Following cyanide exposure, treatment with glyoxylate in both mammalian and non-mammalian animal models confers resistance to cyanide toxicity with greater efficacy and faster kinetics than known cyanide scavengers. Glyoxylate-mediated cyanide resistance is accompanied by rapid pyruvate consumption without an accompanying increase in lactate concentration. Lactate dehydrogenase is required for this effect which distinguishes the mechanism of glyoxylate rescue as distinct from countermeasures based solely on chemical cyanide scavenging. Our metabolic data together support the hypothesis that glyoxylate confers survival at least in part by reversing the cyanide-induced redox imbalances in the cytosol and mitochondria. The data presented herein represent the identification of a potential cyanide countermeasure operating through a novel mechanism of metabolic modulation.
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Glioxilatos , Peixe-Zebra , Animais , Cianetos/toxicidade , Mamíferos , Ácido PirúvicoRESUMO
Brain imaging requires mounting of zebrafish larvae in a vertical position, but anesthetized or fixed larvae tend to fall on their sides without external support. Current solution is to manually hold individual larva until liquid agarose solidifies, which is time consuming, labor intensive, and unfriendly to beginners. We developed a method to form larva-shaped slots in agarose gel using a computer numerical controlled manufactured mold. Each slot nearly perfectly fits a larva in its upright position, and larvae can be easily mounted by inserting into the slots. On average, each larva can be mounted in <1 min using this method.
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Encéfalo , Peixe-Zebra , Animais , Larva , NeuroimagemRESUMO
Recent advances in molecular and cellular engineering, such as human cell reprogramming, genome editing, and patient-specific organoids, have provided unprecedented opportunities for investigating human disorders in both animals and human-based models at an improved pace and precision. This progress will inevitably lead to the development of innovative drug-screening platforms and new patient-specific therapeutics. In this review, we discuss recent advances that have been made using zebrafish and human-induced pluripotent stem cell (iPSC)-derived neurons and organoids for modeling genetic epilepsies. We also provide our prospective on how these models can potentially be combined to build new screening platforms for antiseizure and antiepileptogenic drug discovery that harness the robustness and tractability of zebrafish models as well as the patient-specific genetics and biology of iPSC-derived neurons and organoids.
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Anticonvulsivantes/uso terapêutico , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Epilepsia/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Organoides/fisiologia , Animais , Anticonvulsivantes/farmacologia , Epilepsia/diagnóstico , Epilepsia/tratamento farmacológico , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Organoides/efeitos dos fármacos , Peixe-ZebraRESUMO
CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we introduce Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. The platform can efficiently identify genes responsible for morphological or behavioral phenotypes. In one application, we showed that MIC-Drop could identify small-molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, we discovered several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse genetic screens in model organisms.
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Sistemas CRISPR-Cas , Testes Genéticos , Técnicas Analíticas Microfluídicas , Peixe-Zebra/genética , Animais , Sistema Cardiovascular/crescimento & desenvolvimento , Técnicas de Cultura de Células , Sequenciamento de Nucleotídeos em Larga Escala , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
In our efforts to discover new drugs to treat pain, we identified molleamines A-E (1-5) as major neuroactive components of the sea slug, Pleurobranchus forskalii, and their prey, Didemnum molle, tunicates. The chemical structures of molleamines were elucidated by spectroscopy and confirmed by the total synthesis of molleamines A (1) and C (3). Synthetic 3 completely blocked acetylcholine-induced calcium flux in peptidergic nociceptors (PNs) in the somatosensory nervous system. Compound 3 affected neither the α7 nAChR nor the muscarinic acetylcholine receptors in calcium flux assays. In addition to nociceptors, 3 partially blocked the acetylcholine-induced calcium flux in the sympathetic nervous system, including neurons from the superior cervical ganglion. Electrophysiology revealed a block of α3ß4 (mouse) and α6/α3ß4 (rat) nicotinic acetylcholine receptors (nAChRs), with IC50 values of 1.4 and 3.1 µM, respectively. Molleamine C (3) is a partial antagonist, reaching a maximum block of 76-82% of the acetylcholine signal and showing no partial agonist response. Molleamine C (3) may thus provide a lead compound for the development of neuroactive compounds with unique biological properties.
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Receptores Nicotínicos , Urocordados , Animais , Aplysia , Camundongos , Antagonistas Nicotínicos/farmacologia , Nylons , Ratos , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Opioid use disorder (OUD) has become a leading cause of death in the United States, yet current therapeutic strategies remain highly inadequate. To identify potential treatments for OUD, we screened a targeted selection of over 100 drugs using a recently developed opioid self-administration assay in zebrafish. This paradigm showed that finasteride, a steroidogenesis inhibitor approved for the treatment of benign prostatic hyperplasia and androgenetic alopecia, reduced self-administration of multiple opioids without affecting locomotion or feeding behavior. These findings were confirmed in rats; furthermore, finasteride reduced the physical signs associated with opioid withdrawal. In rat models of neuropathic pain, finasteride did not alter the antinociceptive effect of opioids and reduced withdrawal-induced hyperalgesia. Steroidomic analyses of the brains of fish treated with finasteride revealed a significant increase in dehydroepiandrosterone sulfate (DHEAS). Treatment with precursors of DHEAS reduced opioid self-administration in zebrafish in a fashion akin to the effects of finasteride. These results highlight the importance of steroidogenic pathways as a rich source of therapeutic targets for OUD and point to the potential of finasteride as a new treatment option for this disorder.
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Inibidores de 5-alfa Redutase/farmacologia , Finasterida/farmacologia , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Animais , Modelos Animais de Doenças , Humanos , Masculino , Transtornos Relacionados ao Uso de Opioides/fisiopatologia , Ratos , Ratos Sprague-Dawley , Peixe-ZebraRESUMO
Natural products such as conotoxins have tremendous potential as tools for biomedical research and for the treatment of different human diseases. Conotoxins are peptides present in the venoms of predatory cone snails that have a rich diversity of pharmacological functions. One of the major bottlenecks in natural products research is the rapid identification and evaluation of bioactive molecules. To overcome this limitation, we designed a set of light-induced behavioral assays in zebrafish larvae to screen for bioactive conotoxins. We used this screening approach to test several unique conotoxins derived from different cone snail clades and discovered that a conorfamide from Conus episcopatus, CNF-Ep1, had the most dramatic alterations in the locomotor behavior of zebrafish larvae. Interestingly, CNF-Ep1 is also bioactive in several mouse assay systems when tested in vitro and in vivo. Our novel screening platform can thus accelerate the identification of bioactive marine natural products, and the first compound discovered using this assay has intriguing properties that may uncover novel neuronal circuitry.
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Larva/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Venenos de Moluscos/farmacologia , Neuropeptídeos/farmacologia , Peixe-Zebra , Animais , Caramujo Conus/química , Feminino , Masculino , CamundongosRESUMO
Primordial germ cells (PGCs) are the precursor cells that form during early embryogenesis and later differentiate into oocytes or spermatozoa. Abnormal development of PGCs is frequently a causative factor of infertility and germ cell tumors. However, our understanding of PGC development remains insufficient, and we have few pharmacological tools for manipulating PGC development for biological study or therapy. The zebrafish (Danio rerio) embryos provide an excellent in vivo animal model to study PGCs, because zebrafish embryos are transparent and develop outside the mother. Importantly, the model is also amenable to facile chemical manipulations, including scalable screening to discover novel compounds that alter PGC development. This chapter describes methodologies for manipulating the germline (i.e., PGCs) with small molecules and for monitoring PGC development. Utilizing the 3'UTR of PGC marker genes such as nanos3 and ddx4/vasa is a key component of these methodologies, which consist of expressing fluorescent or luminescent proteins in PGCs, treatment with small molecules, and quantitative observation of PGC development.
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Células Germinativas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Regiões 3' não Traduzidas/genética , Animais , RNA Helicases DEAD-box/genética , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Feminino , Proteínas Luminescentes/genética , Masculino , Proteínas de Ligação a RNA/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Fermentation of Acremonium tubakii W. Gams isolated from a soil sample collected from the University of Utah led to the isolation and characterization of six new linear pentadecapeptides, emerimicins V-X (1-6). Peptaibols containing 15-residues are quite rare, with only 22 reported. Genome mining and bioinformatic analysis were used to identify the emerimicin 60 kbp eme biosynthetic cluster harboring a single 16-module hybrid polyketide-nonribosomal peptide synthetase. A detailed bioinformatic investigation of the corresponding 15 adenylation domains, combined with 1D and 2D NMR experiments, LC-MS/MS data, and advanced Marfey's method, allowed for the elucidation and absolute configuration of all proteinogenic and nonproteinogenic amino acid residues in 1-6. As some peptaibols possess cytotoxic activity, a zebrafish embryotoxicity assay was used to evaluate the toxicity of the six emerimicins and showed that emerimicin V (1) and VI (2) exhibit the most potent activity. Additionally, out of the six emerimicins, 1 displayed modest activity against Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium with MIC values of 64, 32, and 64 µg/mL, respectively.